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  • Here we found that these leukotriene antagonists also


    Here we found that these leukotriene antagonists also inhibit the effects of nucleotides acting at P2Y receptors in dU937 cells, which are known to express a number of nucleotide receptors, such as P2Y2 or P2Y4[30]. In an effort to characterize the P2 receptor subtypes subject to negative modulation by CysLT1 receptor antagonists we studied the interactions of these compounds in human P2Y1, P2Y2, P2Y4, and P2Y6 receptors stably expressed in 1321N1 astrocytoma cells. The latter have been chosen since these cells are devoid of endogenous P2Y receptors and for this reason have been widely utilized for transfecting and characterizing various P2Y receptor subtypes [31], [32].
    Materials and methods
    Discussion A relationship between UDP and the CysLT1 receptor has already been suggested, with the hypothesis that UDP may act directly on the receptor [42], [43]. Nevertheless, the concentrations of CysLT1 antagonists PX-478 2HCl synthesis required to functionally antagonize the effects of UDP in this study are not comparable to the nM concentrations at which the compounds act at the CysLT1 receptor. Although it does not establish the molecular basis for interaction, this study expands the range of possible interactions to other uracil and PX-478 2HCl synthesis nucleotides. In this study, montelukast and pranlukast were found to inhibit nucleotide-induced calcium mobilization in a human monocyte-macrophage like cell line, DMSO-differentiated U937 (dU937) in an insurmountable manner. dU937 cells were shown to express P2Y2, P2Y4, P2Y6, P2Y11, P2Y13 and P2Y14 receptors. Therefore, these antagonists were studied functionally in a heterologous expression system for the human P2Y receptors. In 1321N1 astrocytoma cells stably expressing hP2Y1,2,4,6 receptors, CysLT1 antagonists inhibited both the P2Y agonist-induced activation of phospholipase C and intracellular Ca2+ mobilization. The inhibition was concentration-dependent but in an insurmountable manner. In control astrocytoma cells expressing an endogenous M3 muscarinic receptor, 10μM montelukast had no effect on the carbachol-induced rise in intracellular Ca2+. Thus, these data demonstrated that CysLT1 receptor antagonists interact functionally with signaling pathways of P2Y receptors, and independent of another PLC-coupled receptor. Furthermore, ligand binding studies using [3H]LTD4 excluded the possibility of competitive nucleotide binding to the CysLT1 receptor in dU937 cells, but not eliminating the possibility of an allosteric interaction at the CysLT1 receptor level. Another possible explanation for these effects is that the leukotriene antagonists are interacting directly with the P2Y receptors, to which they are related in the phylogenetic dendrogram of GPCRs [15]. However, in the P2Y1 receptor-expressing astrocytoma cells the binding of a specific antagonist (nucleotide) radioligand of the P2Y1 receptor, [3H]MRS2279, was not displaced by the leukotriene antagonists. Since MRS2279 is a competitive ligand, if the interaction of the leukotriene antagonist is directly with this receptor, it would necessarily be through an allosteric interaction as already suggested by data obtained in dU937 cells (see Fig. 4, Fig. 5). Other examples of allosteric modulation of P2Y receptors have been reported [35]. Another possibility is that the antagonism occurs at a yet unidentified signaling step subsequent to activation of P2Y, but not M3 muscarinic, receptors. Thus, the functional inhibition of the nucleotide-elicited effects was either through direct allosteric interaction with the receptor or through a signaling pathway common to the P2Y receptors but not characteristic of cholinergic signaling. Furthermore, we have recently shown that activation of P2Y receptors with extracellular nucleotides induced heterologous desensitization of the CysLT1 receptor in dU937 cells, but not receptor trafficking or internalization. Conversely, LTD4-induced CysLT1 receptor activation had no effect on P2Y receptor responses, suggesting that the latter have a hierarchy in producing desensitizing signals [14]. Thus, while a putative heterodimerization between CysLT1 and P2Y receptors might as well explain our results, it is unlikely to occur in our system, considering the lack of cross-desensitization, the different mechanism of agonist- or nucleotides-induced CysLT1 desensitization, and the different trafficking induced by LTD4 or by extracellular nucleotides signals [14].